Pramlintide pro H-amylin salts and compositions

ABSTRACT

Agonist analogues of amylin and related pharmaceutical compositions, and methods of treatment of diabetes and other insulin-requiring states, as well as methods of treatment of hypoglycemia, are provided.

This is a division of application Ser. No. 08/447,849 filed May 23, 1995U.S. Pat. No. 5,686,411, which is a continuation of application Ser. No.07/794,266 filed Nov. 19, 1991 (Abn), which is a continuation-in-part ofapplication Ser. No. 07/667,040 filed Mar. 8, 1991 (ABN), which priorapplications are hereby incorporated by reference in their totalities(including drawings).

BACKGROUND

1. Field of the Invention

The field of the invention is medicine, particularly the treatment andprevention of hypoglycemic conditions and other conditions in whichenhanced amylin action is of benefit, including insulin-requiring statessuch as diabetes mellitus. More specifically, the invention relates tothe preparation and use of agonist analogues of the peptide hormoneamylin.

2. Description of Related Art and Introduction to the Invention

Diabetes mellitus is a serious metabolic disease that is defined by thepresence of chronically elevated levels of blood glucose(hyperglycemia). This state of hyperglycemia is the result of a relativeor absolute lack of activity of the peptide hormone, insulin. Insulin isproduced and secreted by the β cells of the pancreas. Insulin isreported to promote glucose utilization, protein synthesis, and theformation and storage of neutral lipids. Glucose, the principal sourceof carbohydrate energy, is stored in the body as glycogen, a form ofpolymerized glucose, which may be converted back into glucose to meetmetabolism requirements. Under normal conditions, insulin is secreted atboth a basal rate and at enhanced rates following glucose stimulation,all to maintain metabolic homeostasis by the conversion of glucose intoglycogen.

The term diabetes mellitus encompasses several different hyperglycemicstates. These states include Type 1 (insulin-dependent diabetes mellitusor IDDM) and Type 2 (non-insulin-dependent diabetes mellitus or NIDDM)diabetes. The hyperglycemia present in individuals with Type I diabetesis associated with deficient, reduced, or nonexistent levels of insulinwhich are insufficient to maintain blood glucose levels within thephysiological range. Treatment of Type 1 diabetes involvesadministration of replacement doses of insulin, generally by theparenteral route. The hyperglycemia present in individuals with Type IIdiabetes is initially associated with normal or elevated levels ofinsulin; however, these individuals are unable to maintain metabolichomeostasis due to a state of insulin resistance in peripheral tissuesand liver and, as the disease advances, due to a progressivedeterioration of the pancreatic β cells which are responsible for thesecretion of insulin. Thus, initial therapy of Type 2 diabetes may bebased on diet and lifestyle changes augmented by therapy with oralhypoglycemic agents such as sulfonylureas. Insulin therapy is oftenrequired, however, especially in the latter stages of the disease, inattempting to produce some control of hyperglycemia and minimizecomplications of the disease. Thus, many Type 2 diabetics ultimatelyrequire insulin in order to survive.

Amyloid is the name given to extracellular deposits of β sheet proteinfilaments. Deposits of amyloid material have been reported to be foundin pancreas of patients with Type 2 diabetes mellitus. Other studieshave indicated that the degree of amyloid depositions increases with thedegree of hyperglycemia in humans and the severity of Type 2 diabetes.Chemical analysis of pancreatic amyloid led to the surprising andunexpected discovery of the peptide hormone, amylin. Clark, A., et al.,Lancet ii: 231-234 (1987). This peptide was discovered to be comprisedof 37 amino acids, none of which are acidic residues, to have adisulfide linkage between the cysteine residues at positions 2 and 7,and to be C-terminally amidated. Amylin is the major protein constituentof the amyloid which is reported to be found in the pancreatic Islets ofLangerhans in patients with type 2 diabetes mellitus.

It has been reported that the presence of both the intramolecularcystine bridge and the carboxy terminal amide group in the peptidestructure of the synthetic molecule yield the greatest biologicalactivity to inhibit glycogen synthesis in skeletal muscle. E.g., Cooper,G. J. S., et al., Proc. Natl. Acad. Sci. (USA) 84:8628-8632 (1987);Cooper G. J. S., et al., in Diabetes 1988, ed. Larkins, R., Zimmet, P. &Chisholm, D. (Elsevier, Amsterdam), pp. 493-496 (1989). The amino acidsequence of amylin (see FIG. 1) has 46% homology with human calcitoningene related peptide 2 (CGRP-2).

One report states that a limited segment of the amylin molecule,residues 20-29, is a potential contributor toward amyloid fibrilformation in the islets of Langerhans in Type 2 diabetes mellitus.Glenner et al., Biochem. Biophys. Res Commun. 155:608-614 (1988). It hasalso been reported that amino acid sequence differences between amylinsfrom certain mammalian species occur in this region, and furtherinvestigation has focused on identifying residues linked to amyloidformation. Westermark et al., Proc. Natl. Acad. Sci. (USA) 87: 5036-5040(1990). The study of Westermark et al. reported attempts to synthesizevarious 20-29 amino acid segments of amylin sequences from differentspecies followed by a comparison of their ability to form amyloidfibrils. It was proposed that the residues 25-29 of human amylin werethe most strongly amyloidogenic and that the proline-for-serinesubstitution in position 28, as in several rodent species, significantlyinhibited fibril formation in the studied decapeptides.

Amylin is a complex peptide, and the synthesis of bioactive preparationsof amylin is laborious. Amylin has also been found to have Limitedsolubility and limited stability in solution. We have found that ratamylin has a higher solubility and stability in solution than humanamylin. This may be due in some measure, although this is not known, tothe different aggregation properties of the amylins from differentspecies. Only the human, non-human primate, and cat species of amylinhave been reported to aggregate to form islet amyloid in vivo. Thesequences of amylin now reported to have been isolated from a number ofspecies are set forth in FIG. 2.

In Type I diabetes, amylin levels are severely reduced or arenonexistent when compared to normal controls. In the disease state ofType I diabetes mellitus, the β-cells, which are the producers ofinsulin and amylin, have been destroyed by an autoimmune process. Amylinhas been proposed to be useful in the treatment of diabetes mellitus andhypoglycemia, including insulin-induced hypoglycemia. It has also beenproposed that the coadministration of insulin with amylin is a superiortherapy to the existing administration of insulin alone, and thatcoadministration of amylin with glucagon for the treatment ofhypoglycemia is a superior therapy to the existing administration ofglucagon alone. It would be useful to provide, for such purposes andothers, less complicated compounds that have the activities of nativehuman amylin, as well as compounds which may show enhanced solubilityand/or stability over native human amylin. Such compounds are describedand claimed herein.

SUMMARY OF THE INVENTION

The present invention is directed to novel analogues of 0the peptidehormone amylin. These compounds mimic the effects of amylin, and arereferred to as amylin agonists or as agonist analogues of amylin.

The invention is also directed to pharmaceutical compositions comprisingthe agonist analogues of the present invention, and to methods oftreatment and prevention of hypoglycemic conditions and other conditionsin which enhanced amylin action is of benefit, includinginsulin-requiring states such as diabetes mellitus, comprisingadministering an agonist analogue of amylin to an animal (alone or inconjunction with an insulin or a glucagon).

Definitions

As used herein, the following terms have the following meanings unlessexpressly stated to the contrary:

The term "alkyl" refers to both straight- and branched-chain alkylgroups. The term "lower alkyl" refers to both straight- andbranched-chain alkyl groups having a total of from 1 to 6 carbon atomsand includes primary, secondary and tertiary alkyl groups. Typical loweralkyls include, for example, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like.

The term "aryl" refers to carbocyclic aromatic groups of 6 to 14 carbonatoms such as phenyl and naphthyl, as well as heterocyclic aromaticgroups containing 1 to 3 heteroatoms (nitrogen, oxygen, sulfur, etc.)such as pyridyl, triazolopyrazine, pyrimidine and the like.

The term "aralkyl" refers to an "aryl" group of 6 to 10 carbon atomsdirectly attached to an "alkyl" group of 1 to 4 carbon atoms andincludes for example benzyl, p-chlorobenzyl, p-methylbenzyl, and2-phenylethyl.

The term "cycloalkyl" refers to cyclic alkyl groups of 5 to 8 carbonatoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of human amylin [SEQ. ID. NO. 1].

FIG. 2 depicts a comparison of amino acid sequences of amylins isolatedfrom several mammals [SEQ. ID. NO. 1-6].

FIG. 3 depicts the amino acid sequence of novel amylin agonist peptides.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, novel agonist analogues of amylinare provided. These analogues are useful as agonists of amylin,including as hyperglycemics, and may be represented by FIG. 3.

In one aspect, the present invention is directed to agonist analogues ofFIG. 3, wherein A₁ is hydrogen Lys, Ser, Ala, des-α-amino Lys, oracetylated Lys; B₁ is Ala, Ser or Thr; C₁ is Val, Leu or Ile; D₁ is Hisor Arg; E₁ is Ser or Thr; F₁ is Ser, Thr, Gln or Asn; G₁ is Asn, Gln orHis; H₁ is Phe, Leu or Tyr; I₁ is Ala or Pro; J₁ is Ile, Val, Ala orLeu; K₁ is Ser, Pro, Leu, Ile or Thr; L₁ is Ser, Pro or Thr; M₁ is Asn,Asp or Gln; X and Y are independently selected residues having sidechains which are chemically bonded to each other to form anintramolecular linkage; and Z is hydroxy, amino, alkylamino,dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy,aryloxy or aralkyloxy; provided that (a) when A₁ is Lys, B₁ is Ala, C₁is Val, D₁ is His, E₁ is Ser, F₁ is Ser, G₁ is Asn, H₁ is Phe, I₁ isAla, J₁ is Ile, K₁ is Ser, L₁ is Ser, and M₁ is Asn; (b) when A₁ is Lys,B₁ is Ala, C₁ is Ile, D₁ is Arg, E₁ is Ser, F₁ is Ser, G₁ is Asn, H₁ isLeu, I₁ is Ala, J₁ is Ile, K₁ is Ser, L₁ is Pro, and M₁ is Asn; (c) whenA₁ is Lys, B₁ is Ala, C₁ is Val, D₁ is Arg, E₁ is Thr, F₁ is Ser, G₁ isAsn, H₁ is Leu, I₁ is Ala, J₁ is Ile, K₁ is Ser, L₁ is Pro, and M₁ isAsn; (d) when A₁ is Lys, B₁ is Ala, C₁ is Val, D₁ is Arg, E₁ is Ser, F₁is Ser, G₁ is Asn, H₁ is Leu, I₁ is Pro, J₁ is Val, K₁ is Pro, L₁ isPro, and M₁ is Asn; (e) when A₁ is Lys, B₁ is Ala, C₁ is Val, D₁ is His,E₁ is Ser, F₁ is Asn, G₁ is Asn, H₁ is Leu, I₁ is Pro, J₁ is Val, K₁ isSer, L₁ is Pro and M₁ is Asn; or (f) when A₁ is Lys, B₁ is Thr, C₁ isVal, D₁ is Arg, E₁ is Ser, F₁ is Ser, G₁ is His, H₁ is Leu, I₁ is Ala,J₁ is Ala, K₁ is Leu, L₁ is Pro and M₁ is Asp; then one or more of anyof A₁ to M₁ is not an L-amino acid and Z is not amino.

Suitable side chains for X and Y include groups derived from alkylsulfhydryls which may form disulfide bonds; alkyl acids and alkyl amineswhich may form cyclic lactams; alkyl aldehydes or alkyl halides andalkylamines which may condense and be reduced to form an alkyl aminebridge; or side chains which may be connected to form an alkyl, alkenyl,alkynyl, ether or thioether bond. Preferred alkyl chains include loweralkyl groups having from about 1 to about 6 carbon atoms.

An additional aspect of the present invention is directed to agonistanalogues of FIG. 3 which are not bridged, and wherein X and Y areindependently selected from Ala, Ser, Cys, Val, Leu and Ile or alkyl,aryl, or aralkyl esters and ethers of Ser or Cys.

Biologically active derivatives of the above FIG. 3 agonist analoguesare also included within the scope of this invention in which thestereochemistry of individual amino acids may be inverted from (L)/S to(D)/R at one or more specific sites.

Also included within the scope of this invention are the agonistanalogues modified by glycosylation of Asn, Ser and/or Thr residues.

Biologically active agonist analogues of amylin are included within thescope of this invention which contain less peptide character. Suchpeptide mimetics may include, for example, one or more of the followingsubstitutions for --CO--NH-- amide bonds: depsipeptides (--CO--O--),iminomethylenes (--CH₂ --NH--), trans-alkenes (--CH═CH--),β-enaminonitriles (--C(═CH--CN)--NH--), thioamides (--CS--NH--),thiomethylenes (--S--CH₂ -- or --CH₂ --S--), methylenes (--CH₂ --CH₂ --)and retro-amides (--NH--CO--).

Compounds of this invention form salts with various inorganic andorganic acids and bases. Such salts include salts prepared with organicand inorganic acids, for example, HCl, HBr, H₂ SO₄, H₃ PO₄,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid,toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonicacid. Salts prepared with bases include, for example, ammonium salts,alkali metal salts (such as sodium and potassium salts) and alkali earthsalts (such as calcium and magnesium salts). Acetate, hydrochloride, andtrifluoroacetate salts are preferred.

The salts may be formed by conventional means, as by reacting the freeacid or base forms of the product with one or more equivalents of theappropriate base or acid in a solvent or medium in which the salt isinsoluble, or in a solvent such as water which is then removed in vacuoor by freeze-drying or by exchanging the ions of an existing salt foranother ion on a suitable ion exchange resin.

The compounds of the invention include various stereoisomers. In thepreferred compounds of this invention, the chiral centers on the peptidebackbone are all S.

Compounds of the present invention may be prepared by using certainconventional coupling reactions known in the peptide art. The analoguesof this invention are prepared by successively adding the desired aminoacid to a growing peptide chain. Typically, an α-N-carbamoyl protectedamino acid and an amino acid attached to the growing peptide chain on aresin support are reacted at room temperature in an inert solvent suchas N-methylpyrrolidone, dimethylformamide or methylene chloride in thepresence of coupling agents such as dicyclohexylcarbodiimide1-hydroxybenzotriazole in the presence of a base such asdiisopropylethylamine. The α-N-carbamoyl protecting group is removedfrom the resultant peptide with a reagent such as trifluoroacetic acidor piperidine, and the coupling reaction repeated with the next desiredN-protected amino acid. Suitable N-protecting groups are known in theart, with t-butyloxycarbonyl herein preferred.

Certain preferred methods for synthesis are described in thecommonly-assigned copending and commonly assigned patent applicationSer. No. 667,040 ("Synthetic Preparation of Amylin and Amylin Analogs",filed Mar. 8, 1991) (ABN). These methods provide for solid phasesynthesis of a peptide which comprises amylin or an amylin analog whichhas enhanced biological activity and is substantially free of deletionand other contaminating peptides wherein said peptide is synthesizedusing successive synthesis cycles, whereby in each such synthesis cycle,a designated amino acid is added to a growing peptide chain attached toan insoluble resin support by formation of a peptide linkage between anα-amino group of the growing peptide chain and on α-carboxyl of thedesignated amino acid; and wherein each synthesis cycle comprises: (a)treating the growing peptide chain under α-amino deprotecting conditionsto remove an α-amino group; (b) activating the α-carboxyl group of theα-amino protected designated amino acid; (c) contacting the growingpeptide chain and the designated amino acid under coupling conditions toform a peptide linkage between the free α-amino for the peptide chainand the activated α-carboxyl of the designated amino acid; and (d)repeating steps (b) and (c) if the coupling efficiency of step (c) isless than about 97%. It is preferred to repeat steps (b) and (c) if thecoupling efficiency is less than about 99%. In another preferred aspect,steps (b) and (c) are repeated in each synthesis cycle. Optionally, thecoupling efficiency is measured after each coupling step.

Suitable coupling conditions include use of a solvent system whichmaximizes swelling of the solid support, minimizes secondary structureelements of the peptide chain during synthesis cycles, and minimizesintrapeptide and interpeptide hydrogen bonding. Preferably the synthesiscycle includes a capping step after the coupling step(s) whereinunreacted α-amino groups of the peptide chain are rendered unreactive.The synthesis cycle is successively repeated using appropriate protectedα-amino acids to give amylin or an amylin analog of specified sequence.After completions of the successive synthesis cycles, said amylin oramylin analog is cleaved from the solid support. It is preferred thatthe cysteine residues of the peptide chain are selectively deprotectedand an intramolecular disulfide bond is formed before cleaving thepeptide bond from the solid support.

Suitable α-amino protective groups include t-butoxycarbonyl and9-fluorenylmethoxycarbonyl. In one preferred aspect, whent-butoxycarbonyl is used as the α-amino protecting group, the α-carboxylgroups are activated using dicyclohexylcarbodiimide and1-hydroxybenzotriazole to form 1-hydroxybenzotriazole esters. Aparticularly preferred solvent system comprises N-methylpyrrolidone.

The preparation of certain agonist analogues of amylin within theinvention is described in Examples 1 to 17 herein. In addition, otheragonist analogues which may be prepared according to the aboveprocedures are set forth in Table II herein. The compounds of theinvention may also be prepared using recombinant DNA techniques, usingmethods now known in the art. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989).

The nomenclature of the compounds of the present invention can be usedto indicate both the peptide that the sequence is based on and themodifications made to any basic peptide amylin sequence, such as humanamylin. An amino acid preceded by a superscript number indicates thatthe named amino acid replaces the amino acid normally present at theamino acid position of the superscript in the basic amino acid sequence.For example, "¹⁸ Arg²⁵,28 Pro-h-amylin" refers to a peptide based on thesequence of "h-amylin" or "human-amylin" having the followingsubstitutions: Arg replacing His at residue 18, Pro replacing Ala atresidue 25 and Pro replacing Ser at residue 28. The term "des-¹Lys-h-amylin" refers to a peptide based on the sequence of human amylin,with the first, or N-terminal, amino acid deleted.

The agonist analogues of amylin of this invention are useful in view oftheir pharmacological properties. In particular, compounds of thisinvention possess activity as amylin agonist agents, as will beevidenced by activity in the receptor binding assay and the soleusmuscle assay described in Examples 18 and 19, respectively. Amylinagonist activity of compounds may also be assessed by the ability toinduce hyperlactemia and/or hyperglycemia in mammals. In addition to thedescription of compounds pursuant to FIG. 3, certain preferred compoundsare set forth in Table I. The preferred compounds des-¹ Lys-h-amylin[SEQ. ID. NO. 7], ²⁸ Pro-h-amylin [SEQ. ID. NO. 8], ²⁵,28,29Pro-h-amylin [SEQ. ID. NO. 9], ¹⁸ Arg²⁵,28 Pro-h-amylin [SEQ. ID. NO.10], and des-¹ Lys¹⁸ Arg²⁵,28 Pro-h-amylin [SEQ. ID. NO. 11], all showamylin activity in vivo in treated test animals, provoking markedhyperlactemia followed by hyperglycemia. In addition to havingactivities characteristic of amylin, certain of the preferred compoundsof the invention have also been found to possess more desirablesolubility and stability characteristics when compared to human amylin.These preferred compounds include ²⁵ Pro²⁶ Val²⁸,29 Pro-h-amylin [SEQ.ID. NO. 12], ²⁵,28,29 Pro-h-amylin, and ¹⁸ Arg²⁵,28 Pro-h-amylin.

Compounds described herein which are especially preferred include ¹⁸Arg²⁵,28 Pro-h-amylin, des-¹ Lys¹⁸ Arg²⁵,28 Pro-h-amylin, ¹⁸ Arg²⁵,28,29Pro-h-amylin [SEQ. ID. NO. 13], des-¹ Lys¹⁸ Arg²⁵,28,29 Pro-h-amylin[SEQ. ID. NO. 14], ²⁵,28,29 Pro-h-amylin, des-¹ Lys²⁵,28,29 Pro-h-amylin[SEQ. ID. NO. 15], and ²⁵ Pro²⁶ Val²⁸,29 Pro-h-amylin. Still furtheramylin agonist peptide compounds are listed in Table II. They include:

²³ Leu²⁵ Pro²⁶ Val²⁸,29 Pro-h-amylin [SEQ. ID. NO. 16];

²³ Leu²⁵ Pro²⁸ Val²⁸ Pro-h-amylin [SEQ. ID. NO. 17];

des-¹ Lys²³ Leu²⁵ Pro²⁶ Val²⁸ Pro-h-amylin [SEQ. ID. NO. 18];

¹⁸ Arg²³ Leu²⁵ Pro²⁶ Val²⁸ Pro-h-amylin [SEQ. ID. NO. 19];

¹⁸ Arg²³ Leu²⁵,28,29 Pro-h-amylin [SEQ. ID. NO. 20];

¹⁸ Arg²³ Leu²⁵,28 Pro-h-amylin [SEQ. ID. NO. 21];

¹⁷ Ile²³ Leu²⁵,28,29 Pro-h-amylin [SEQ. ID. NO. 22];

¹⁷ Ile²⁵,28,29 Pro-h-amylin [SEQ. ID. NO. 23];

des-¹ Lys¹⁷ Ile²³ Leu²⁵,28,29 Pro-h-amylin [SEQ. ID. NO. 24];

¹⁷ Ile¹⁸ Arg²³ Leu-h-amylin [SEQ. ID. NO. 25];

¹⁷ Ile¹⁸ Arg²³ Leu²⁶ Val²⁹ Pro-h-amylin [SEQ. ID. NO. 26];

¹⁷ Ile¹⁸ Arg²³ Leu²⁵ Pro²⁶ Val²⁸, 29Pro -h-amylin [SEQ. ID. NO. 27];

¹³ Thr²¹ His²³ Leu²⁶ Ala²⁸ Leu29Pro³¹ Asp-h-amylin [SEQ. ID. NO. 28];

¹³ Thr²¹ His²³ Leu²⁶ Ala²⁹ Pro³¹ Asp-h-amylin [SEQ. ID. NO. 29];

Des-1Lys13Thr21His23Leu26Ala28Pro31Asp-h-amylin [SEQ. ID. NO. 30];

¹³ Thr¹⁸ Arg²¹ His²³ Leu²⁶ Ala²⁹ Pro³¹ Asp-h-amylin [SEQ. ID. NO. 31];

¹³ Thr¹⁸ Arg²¹ His²³ Leu²⁸,29 Pro³¹ Asp-h-amylin [SEQ. ID. NO. 32]; and,

¹³ Thr¹⁸ Arg²¹ His²³ Leu²⁵ Pro²⁶ Ala²⁸,29 Pro³¹ Asp-h-amylin [SEQ. ID.NO. 33].

The compounds of this invention can be combined with pharmaceuticalcarriers to prepare pharmaceutical forms suitable for parenteraladministration. Experimental responses of the compounds support theclinical application of such pharmaceutical compositions in thetreatment of diabetes mellitus and other insulin-requiring states, aswell as in the prevention and treatment of episodes of hypoglycemia. Thecompounds of this invention can also be combined with insulin for thetreatment of diabetes mellitus and other insulin-requiring states. By"insulin" is meant a polypeptide or its equivalent useful in regulationof blood glucose levels. A general description of such insulins isprovided in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press (1990). Such insulins can be fastacting, intermediate acting, or long acting. Various derivatives ofinsulin exist and are useful in this invention. See, e.g., U.S. Pat.Nos. 5,049,547, 5,028,587, and 5,016,643. Insulin peptides are alsouseful (see, e.g., U.S. Pat. No. 5,008,241), as are analogues (see,e.g., U.S. Pat. Nos. 4,992,417 and 4,992,418). Such compositions can beadministered by any standard route, including nasal administration (see,e.g., U.S. Pat. Nos. 4,988,512 and 4,985,242, and 2 BioWorld Today, No.125 (1991)). The compounds of this invention are also useful incombination with a glucagon for the prevention and treatment ofhypoglycemia. See Young et al., U.S. application Ser. No. 07/640,478(U.S. Pat. No. 5,234,906), filed Jan. 10, 1991, entitled "HyperglycemicCompositions," which is incorporated herein by reference.

Compositions or products of the invention may conveniently be providedin the form of solutions suitable for parenteral (including intravenous,intramuscular and subcutaneous) or nasal or oral administration. In manycases, it will be convenient to provide an agonist analogue of amylinand an insulin or glucagon in a single composition or solution foradministration together. In other cases, it may be more advantageous toadminister an insulin or a glucagon separately from said agonistanalogue. A suitable administration format may best be determined by amedical practitioner for each patient individually. Suitablepharmaceutically acceptable carriers and their formulation are describedin standard formulation treatises, e.g., Remington's PharmaceuticalSciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A."Parenteral Formulations of Proteins and Peptides: Stability andStabilizers," Journal of Parenteral Science and Technology, TechnicalReport No. 10, Supp. 42:2S (1988). Suitable formulations includinginsulin or glucagon are known in the art.

The agonist preparations of the invention may be stabilized at neutralpH. Since the products of the invention are amphoteric they may beutilized as free bases, as acid addition salts or as metal salts. Thesalts must, of course, be pharmaceutically acceptable, and these willinclude metal salts, particularly alkali and alkaline earth metal salts,e.g., potassium or sodium salts. A wide variety of pharmaceuticallyacceptable acid addition salts are available, as described above. Theseinclude those prepared from both organic and inorganic acids, preferablymineral acids. Typical acids which may be mentioned by way of exampleinclude citric, succinic, lactic, hydrochloric and hydrobromic acids.Such products are readily prepared by procedures well known to thoseskilled in the art.

The products of the invention will normally be provided as parenteralcompositions for injection or infusion. They can, for example, besuspended in an inert oil, suitably a vegetable oil such as sesame,peanut, or olive oil. Alternatively, they can be suspended in an aqueousisotonic buffer solution at a pH of about 5.6 to 7.4. Useful buffersinclude sodium citrate-citric acid and sodium phosphate-phosphoric acid.A form of repository or "depot" slow release preparation may be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection.

The desired isotonicity may be accomplished using sodium chloride orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. Sodium chloride ispreferred particularly for buffers containing sodium ions.

If desired, solutions of the above compositions may be thickened with athickening agent such as methylcellulose. They may be prepared inemulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

The therapeutically useful compositions of the invention are prepared bymixing the ingredients following generally accepted procedures. Forexample, the selected components may be simply mixed in a blender orother standard device to produce a concentrated mixture which may thenbe adjusted to the final concentration and viscosity by the addition ofwater or thickening agent and possibly a buffer to control pH or anadditional solute to control tonicity.

For use by the physician, the compositions will be provided in dosageunit form containing an amount of an agonist compound with or withoutinsulin or glucagon which will be effective in one or multiple doses tocontrol or reestablish blood sugar at the selected level.Therapeutically effective amounts of an agonist analogue of amylin asdescribed herein for the treatment of hypoglycemia are those thatincrease blood sugar levels, preferably to above 80 mg/dl.Therapeutically effective amounts of such agonist analogues for thetreatment of diabetes mellitus and other insulin-requiring states arethose sufficient to provide for reduced incidence of insulin overdose orundesired hypoglycemia. As will be recognized by those in the field, aneffective amount of therapeutic agent will vary with many factorsincluding the age and weight of the patient, the patient's physicalcondition, the blood sugar level to be obtained, and other factors.Typical dosage units for treatment of diabetes mellitus will containfrom about 0.1 to 5 mg of an amylin agonist compound and, if desired,about 0.5 to about 10 mg of an insulin. Typical dosage units for thetreatment of hypoglycemia will contain about 0.5 to 1.0 mg of an amylinagonist compound and, if desired, the art recognized quantity, or less,of a glucagon.

As set forth above, compositions useful in the invention are formulatedby standard procedure. These compositions are also administered bystandard procedure. Suitable doses are readily determined by those inthe art, examples of which are provided above.

To assist in understanding the present invention, the following examplesare included which describe the results of a series of experiments. Thefollowing examples relating to this invention should not, of course, beconstrued as specifically limiting the invention. Such variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the present invention as hereinafter claimed.

EXAMPLES Example 1 Preparation of ²⁸ Pro-human-Amylin [SEQ. ID. NO. 8]

Solid phase synthesis of this analogue of human ("h-") amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid hydrofluoric acid("HF") in the presence of dimethylsulfide and anisole. The ²⁸Pro-h-amylin was purified by preparative HPLC. The peptide was found tobe homogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+1)/e=3914.

Example 2 Preparation of ²⁵ Pro²⁶ Val²⁸,29 Pro-h-Amylin [SEQ. ID. NO.12]

Solid phase synthesis of this amylin analogue usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment withthallium (III) trifluoroacetate in trifluoroacetic acid. Aftercyclization was achieved the resin and side chain protecting groups werecleaved with liquid HF in the presence of dimethylsulfide and anisole.The ²⁵ Pro²⁶ Val²⁸,29 Pro-h-amylin was purified by preparative HPLC. Thepeptide was found to be homogeneous by analytical HPLC and capillaryelectrophoresis and the structure confirmed by amino acid analysis andsequence analysis. The product gave the desired mass ion. FAB mass spec:(M+1)/e=3936.

Example 3 Preparation of ²,7 Cyclo-[² Asp,⁷ Lys]-h-Amylin [SEQ. ID. NO.34]

Solid phase synthesis of this amylin analogue usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. ² Aspand ⁷ Lys were introduced with Boc-² Asp(Fmoc)-OH and Boc-⁷ Lys(Fmoc)-OH. Following selective side-chain deprotection with piperidinethe side-chain to side-chain (² Asp-⁷ Lys) cyclization was carried outusing Benzotriazol-1yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP reagent). Cyclization was as described in DiMaio, J., et al,. J. Med. Chem. 33:661-667 (1990); Felix, A. M., et al.,Int J. Pept. Prot. Res. 32:441 (1988). The ²,7 cyclo-[² Asp,⁷Lys]amylin-MBHA-resin obtained after cyclization was cleaved with liquidHF in the presence of dimethylsulfide and anisole. The ²,7 cyclo-[²Asp,⁷ Lys]-h-amylin was purified by preparative HPLC. The peptide wasfound to be homogeneous by analytical HPLC and capillary electrophoresisand the structure confirmed by amino acid analysis and sequenceanalysis. FAB mass spec: (M+1)/e=3925.

Example 4 Preparation of des-¹ Lys-h-Amylin [SEQ. ID. NO. 7]

Solid phase synthesis of des-¹ Lys-h-amylin (also represented as ²⁻³⁷h-amylin) using methylbenzhydrylamine anchor-bond resin and N^(a)-Boc/benzyl-side chain protection was carried out by standard peptidesynthesis methods. The ²,7 -[disulfide]amylin-MBHA-resin was obtained bytreatment of Acm-protected cysteines with thallium (III)trifluoroacetate in trifluoroacetic acid. After cyclization was achievedthe resin and side chain protecting groups were cleaved with liquid HFin the presence of dimethylsulfide and anisole. The des-¹ Lys-h-amylinwas purified by preparative reversed-phase HPLC. The peptide was foundto be homogeneous by analytical HPLC and capillary electrophoresis andthe structure confirmed by amino acid analysis and sequence analysis.The product gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,775.

Example 5 Preparation of ¹ Ala-h-Amylin [SEQ. ID. NO. 35]

Solid phase synthesis of ¹ Ala-h-amylin using methylbenzhydrylamineanchor-bond resin and N^(a) -Boc/benzyl-side chain protection wascarried out by standard peptide synthesis methods. The ²,7-[disulfide]amylin-MBHA-resin was obtained by treatment of Acm-protectedcysteines with thallium (III) trifluoroacetate in trifluoroacetic acid.After cyclization was achieved the resin and side chain protectinggroups were cleaved with liquid HF in the presence of dimethylsulfideand anisole. The ¹ Ala-h-amylin was purified by preparativereversed-phase HPLC. The peptide was found to be homogeneous byanalytical HPLC and capillary electrophoresis and the structureconfirmed by amino acid analysis and sequence analysis. The product gavethe desired mass ion. FAB mass spec: (M+H)⁺ =3,847.

Example 6 Preparation of ¹ Ser-h-Amylin [SEQ. ID. NO. 36]

Solid phase synthesis of ¹ Ser-h-amylin using methylbenzhydrylamineanchor-bond resin and N^(a) -Boc/benzyl-side chain protection wascarried out by standard peptide synthesis methods. The ²,7-[disulfide]amylin-MBHA-resin was obtained by treatment of Acm-protectedcysteines with thallium (III) trifluoroacetate in trifluoroacetic acid.After cyclization was achieved the resin and side chain protectinggroups were cleaved with liquid HF in the presence of dimethylsulfideand anisole. The ¹ Ser-h-amylin was purified by preparativereversed-phase HPLC. The peptide was found to be homogeneous byanalytical HPLC and capillary electrophoresis and the structureconfirmed by amino acid analysis and sequence analysis. The product gavethe desired mass ion. FAB mass spec: (M+H)⁺ =3,863.

Example 7 Preparation of ²⁹ Pro-h-Amylin [SEQ. ID. NO. 37]

Solid phase synthesis of this analogue of human amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The ²⁹ Pro-h-amylin was purified bypreparative HPLC. The peptide was found to be homogeneous by analyticalHPLC and capillary electrophoresis and the structure confirmed by aminoacid analysis and sequence analysis. The product gave the desired massion. FAB mass spec: (M+H)⁺ =3916.

Example 8 Preparation of ²⁵,28 Pro-h-Amylin [SEQ. ID. NO. 38]

Solid phase synthesis of ²⁵,28 Pro-h-amylin using methylbenzhydrylamineanchor-bond resin and N^(a) -Boc/benzyl-side chain protection wascarried out by standard peptide synthesis methods. The ²,7-[disulfide]amylin-MBHA-resin was obtained by treatment of Acm-protectedcysteines with thallium (III) trifluoroacetate in trifluoroacetic acid.After cyclization was achieved the resin and side chain protectinggroups were cleaved with liquid HF in the presence of dimethylsulfideand anisole. The ²⁵,28 Pro-h-amylin was purified by preparativereversed-phase HPLC. The peptide was found to be homogeneous byanalytical HPLC and capillary electrophoresis and the structureconfirmed by amino acid analysis and sequence analysis. The product gavethe desired mass ion. FAB mass spec: (M+H)⁺ =3,939.

Example 9 Preparation of des-¹ Lys²⁵,28 Pro-h-Amylin [SEQ. ID. NO. 39]

Solid phase synthesis of des-¹ Lys²⁵,28 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The des-¹ Lys²⁵,28 Pro-h-amylin waspurified by preparative reversed-phase HPLC. The peptide was found to behomogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,811.

Example 10 Preparation of ¹⁸ Arg²⁵,28 Pro-h-Amylin [SEQ. ID. NO. 10]

Solid phase synthesis of ¹⁸ Arg²⁵,28 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The ¹⁸ Arg²⁵,28 Pro-h-amylin was purifiedby preparative reversed-phase HPLC. The peptide was found to behomogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,959.

Example 11 Preparation of des-¹ Lys¹⁸ Arg²⁵,28 Pro-h-Amylin [SEQ. ID.NO. 11]

Solid phase synthesis of des-¹ Lys¹⁸ Arg²⁵,28 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The des-¹ Lys¹⁸ Arg²⁵,28 Pro-h-amylin waspurified by preparative reversed-phase HPLC. The peptide was found to behomogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,832.

Example 12 Preparation of ¹⁸ Arg²⁵,28,29 Pro-h-Amylin [SEQ. ID. NO. 13]

Solid phase synthesis of ¹⁸ Arg²⁵,28,29 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The ¹⁸ Arg²⁵,28,29 Pro-h-amylin waspurified by preparative reversed-phase HPLC. The peptide was found to behomogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,971.

Example 13 Preparation of des-¹ Lys¹⁸ Arg²⁵,28,29 Pro-h-Amylin [SEQ. ID.NO. 14]

Solid phase synthesis of des-¹ Lys¹⁸ Arg²⁵,28,29 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The des-¹ Lys¹⁸ Arg²⁵,28,29 Pro-h-amylinwas purified by preparative reversed-phase HPLC. The peptide was foundto be homogeneous by analytical HPLC and capillary electrophoresis andthe structure confirmed by amino acid analysis and sequence analysis.The product gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,843.

Example 14 Preparation of ²⁵,28,29 Pro-h-Amylin [SEQ. ID. NO. 9]

Solid phase synthesis of ²⁵,28,29 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The ²⁵,28,29 Pro-h-amylin was purified bypreparative reversed-phase HPLC. The peptide was found to be homogeneousby analytical HPLC and capillary electrophoresis and the structureconfirmed by amino acid analysis and sequence analysis. The product gavethe desired mass ion. FAB mass spec: (M+H)⁺ =3,949.

Example 15 Preparation of des-¹ Lys²⁵,28,29 Pro-h-Amylin [SEQ. ID. NO.15]

Solid phase synthesis of des-¹ Lys²⁵,28,29 Pro-h-amylin usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection was carried out by standard peptide synthesis methods. The²,7 -[disulfide]amylin-MBHA-resin was obtained by treatment ofAcm-protected cysteines with thallium (III) trifluoroacetate intrifluoroacetic acid. After cyclization was achieved the resin and sidechain protecting groups were cleaved with liquid HF in the presence ofdimethylsulfide and anisole. The des-¹ Lys²⁵,28,29 Pro-h-amylin waspurified by preparative reversed-phase HPLC. The peptide was found to behomogeneous by analytical HPLC and capillary electrophoresis and thestructure confirmed by amino acid analysis and sequence analysis. Theproduct gave the desired mass ion. FAB mass spec: (M+H)⁺ =3,823.

Example 16 Preparation of des-¹ Lys²⁵ Pro²⁶ Val²⁸,29 Pro-h-Amylin [SEQ.ID. NO. 40]

Solid phase synthesis of this h-amylin analogue usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection is carried out by standard peptide synthesis methods, and the²,7 -[disulfide]amylin-MBHA-resin obtained by treatment with thallium(III) trifluoroacetate in trifluoroacetic acid. After cyclization isachieved the resin and side chain protecting groups are cleaved withliquid HF in the presence of dimethylsulfide and anisole. The des-¹Lys²⁵ Pro²⁶ Val²⁸,29 Pro-h-amylin is then purified by preparative HPLC.

Example 17 Preparation of [(D)-¹¹ Arg]-Amylin [SEQ. ID. NO. 41]

Solid phase synthesis of this amylin analogue usingmethylbenzhydrylamine anchor-bond resin and N^(a) -Boc/benzyl-side chainprotection is carried out by standard peptide synthesis methods. (D)-¹¹Arg is introduced with Boc-(D)-¹¹ Arg(Mtr)-OH. The ²,7-[disulfide]amylin-MBHA-resin, obtained by treatment with thallium (III)trifluoroacetate in trifluoroacetic acid, is cyclized and the resin andside chain protecting groups are cleaved with liquid HF in the presenceof dimethylsulfide and anisole. The [(D)-¹¹ Arg]-amylin is then purifiedby preparative HPLC.

Example 18 Receptor Binding Assay

Evaluation of the binding of compounds of the invention to amylinreceptors was carried out as follows. ¹²⁵ I-rat amylin (Bolton-Hunterlabeled at the N-terminal lysine) was purchased from AmershamCorporation (Arlington Heights, Ill.). Specific activities at time ofuse ranged from 1950 to 2000 Ci/mmol. Unlabeled peptides were obtainedfrom BACHEM Inc. (Torrance, Calif.) and Peninsula Laboratories (Belmont,Calif.).

Male Sprague-Dawley rats (200-250) grams were sacrificed bydecapitation. Brains were removed to cold phosphate-buffered saline(PBS). From the ventral surface, cuts were made rostral to thehypothalamus, bounded laterally by the olfactory tracts and extending ata 45Ε angle medially from these tracts. This basal forebrain tissue,containing the nucleus accumbens and surrounding regions, was weighedand homogenized in ice-cold 20 mM HEPES buffer (20 mM HEPES acid, pHadjusted to 7.4 with NaOH at 23 ΕC). Membranes were washed three timesin fresh buffer by centrifugation for 15 minutes at 48,000× g. The finalmembrane pellet was resuspended in 20 mM HEPES buffer containing 0.2 mMphenylmethylsulfonyl fluoride (PMSF).

To measure ¹²⁵ I-amylin binding, membranes from 4 mg original wet weightof tissue were incubated with ¹²⁵ I-amylin at 12-16 pM in 20 mM HEPESbuffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine serum albumin,and 0.2 mM PMSF. Solutions were incubated for 60 minutes at 23 ΕC.Incubations were terminated by filtration through GF/B glass fiberfilters (Whatman Inc., Clifton, N.J.) which had been presoaked for 4hours in 0.3% poylethyleneimine in order to reduce nonspecific bindingof radiolabeled peptides. Filters were washed immediately beforefiltration with 5 ml cold PBS, and immediately after filtration with 15ml cold PBS. Filters were removed and radioactivity assessed in agamma-counter at a counting efficiency of 77%. Competition curves weregenerated by measuring binding in the presence of 10⁻¹² to 10⁻⁶ Munlabeled test compound and were analyzed by nonlinear regression usinga 4-parameter logistic equation (Inplot program; GraphPAD Software, SanDiego).

In this assay, purified human amylin binds to its receptor at a measuredIC₅₀ of about 50 pM. Results for test compounds of the invention are setforth in Table I, showing that each of the compounds has significantreceptor binding activity.

Example 19 Soleus Muscle Assay

Evaluation of the amylin agonist activity of compounds of the inventionwas carried out using the soleus muscle assay as follows. Male HarlanSprague-Dawley rats of approximately 200 g mass were used in order tomaintain mass of the split soleus muscle less than 40 mg. The animalswere fasted for 4 hours prior to sacrifice by decapitation. The skin wasstripped from the lower limb which was then pinned out on corkboard. Thetendo achilles was cut just above os calcis and m. gastrocnemiusreflected out from the posterior aspect of the tibia. M. soleus, a small15-20 mm long, 0.5 mm thick flat muscle on the bone surface of m.gastrocnemius was then stripped clear and the perimysium cleaned offusing fine scissors and forceps. M. soleus was then split into equalparts using a blade passed antero-posteriorly through the belly of themuscle to obtain a total of 4 muscle strips from each animal. Afterdissecting the muscle from the animal, it was kept for a short period inphysiological saline. It was not necessary that the muscle be held undertension as this had no demonstrable effects on radioglucoseincorporation into glycogen.

Muscles were added to 50 mL Erlenmeyer flasks containing 10 mL of apregassed Krebs-Ringer bicarbonate buffer containing (each liter) NaCl118.5 mmol (6.93 g), KCl 5.94 mmol (443 mg), CaCl₂ 2.54 mmol (282 mg),MgSO₄ 1.19 mmol (143 mg), KH₂ PO₄ 1.19 mmol (162 mg), NaHCO₃ 25 mmol(2.1 g), 5.5 mmol glucose (1 g) and recombinant human insulin(Humulin-R, Eli Lilly, IN) and the test compound, as detailed below. pHat 37 ΕC was verified as being between 7.1 and 7.4. Muscles wereassigned to different flasks so that the 4 muscle pieces from eachanimal were evenly distributed among the different assay conditions. Theincubation media were gassed by gently blowing carbogen (95% O₂, 5% CO₂)over the surface while being continuously agitated at 37 ΕC in anoscillating water bath. After a half-hour "preincubation" period, 0.5μCi of U-¹⁴ C-glucose was added to each flask which was incubated for afurther 60 minutes. Each muscle piece was then rapidly removed, blattedand frozen in liquid N₂, weighed and stored for subsequent determinationof ¹⁴ C-glycogen.

¹⁴ C-glycogen determination was performed in a 7 mL scintillation vial.Each frozen muscle specimen was placed in a vial and digested in 1 mL60% potassium hydroxide at 70 ΕC for 45 minutes under continuousagitation. Dissolved glycogen was precipitated out onto the vial by theaddition of 3 mL absolute ethanol and overnight cooling at -20 ΕC. Thesupernatant was gently aspirated, the glycogen washed again withethanol, aspirated and the precipitate dried under vacuum. All ethanolis evaporated to avoid quenching during scintillation counting. Theremaining glycogen was redissolved in 1 mL water and 4 mL scintillationfluid and counted for ¹⁴ C.

The rate of glucose incorporation into glycogen (expressed in μmol/g/hr)was obtained from the specific activity of ¹⁴ C-glucose in the 5.5 mMglucose of the incubation medium, and the total ¹⁴ C counts remaining inthe glycogen extracted from each muscle. Dose/response curves werefitted to a 4-parameter logistic model using a least-squares iterativeroutine (ALLFIT, v2.7, NIH, MD) to derive EC₅₀ 's. Since EC₅₀ islog-normally distributed, it is expressed ∀ standard error of thelogarithm. Pairwise comparisons were performed using t-test basedroutines of SYSTAT (Wilkinson, "SYSTAT: the system for statistics,"SYSTAT Inc., Evanston Ill. (1989)).

Dose response curves were generated with muscles added to mediacontaining 7.1 nM (1000 μU/mL) insulin and each test compound added atfinal (nominal) concentrations of 0, 1, 3, 10, 30, 100, 300 and 1000 nM.Each assay also contained internal positive controls consisting of asingle batch of archived rat amylin, lyophilized and stored at -70 ΕC.

Human amylin is a known hyperglycemic peptide, and EC₅₀ measurements ofamylin preparations in the soleus muscle assay range typically fromabout 1-10 nM, although some commercial preparations which are less than90% pure have higher EC₅₀ 's due to the presence of contaminants thatresult in a lower measured activity. Results for test compounds are setforth in Table I, showing that each of the compounds has amylinactivity.

                  TABLE I                                                         ______________________________________                                                           Receptor                                                                              Soleus                                               Binding Muscle                                                                Assay IC.sub.50 Assay EC.sub.50                                               (pM) (nM)                                                                   ______________________________________                                        1)   .sup.28 Pro-h-Amylin                                                                              15.0      2.64                                         2) .sup.25 Pro.sup.26 Val.sup.28,29 Pro-h-Amylin 18.0 4.68                    3) .sup.2,7 Cyclo-[.sup.2 Asp,.sup.7 Lys]-h-Amylin 310.0 6.62                 4) .sup.2-37 h-Amylin 236.0 1.63                                              5) .sup.1 Ala-h-Amylin 148.0 12.78                                            6) .sup.1 Ser-h-Amylin 33.0 8.70                                              7) .sup.29 Pro-h-Amylin 64.0 3.75                                             8) .sup.25,28 Pro-h-Amylin 26.0 13.20                                         9) des-.sup.1 Lys.sup.25,28 Pro-h-Amylin 85.0 7.70                            10)  .sup.18 Arg.sup.25,28 Pro-h-Amylin 32.0 2.83                             11)  des-.sup.1 Lys.sup.18 Arg.sup.25,28 Pro-h-Amylin 82.0 3.77                                                 12)  .sup.18 Arg.sup.25,28,29                                                Pro-h-Amylin 21.0 1.25                       13)  des-.sup.1 Lys.sup.18 Arg.sup.25,28,29 Pro-h-Amylin 21.0 1.86                                              14)  .sup.25,28,29 Pro-h-Amylin 10.0                                         3.71                                         15)  des-.sup.1 Lys.sup.25,28,29 Pro-h-Amylin 14.0 4.15                     ______________________________________                                    

                                      TABLE II                                    __________________________________________________________________________    A.sub.1 B.sub.1                                                                          C.sub.1                                                                          D.sub.1                                                                          E.sub.1                                                                          F.sub.1                                                                          G.sub.1                                                                          H.sub.1                                                                          I.sub.1                                                                          J.sub.1                                                                          K.sub.1                                                                          L.sub.1                                                                          M.sub.1                                                                          Z                                 __________________________________________________________________________    18)                                                                              Lys  Ala                                                                              Val                                                                              His                                                                              Ser                                                                              Ser                                                                              Asn                                                                              Leu                                                                              Pro                                                                              Val                                                                              Pro                                                                              Pro                                                                              Asn                                                                              --NH.sub.2                          19) Lys Ala Val His Ser Ser Asn Leu Pro Val Pro Ser Asn --NH.sub.2                                                       20) Hydrogen Ala Val His Ser                                                 Ser Asn Leu Pro Val Pro Ser                                                   Asn --NH.sub.2                      21) Lys Ala Val Arg Ser Ser Asn Leu Pro Val Pro Ser Asn --NH.sub.2                                                       22) Lys Ala Val Arg Ser Ser                                                  Asn Leu Pro Ile Pro Pro Asn                                                   --NH.sub.2                          23) Lys Ala Val Arg Ser Ser Asn Leu Pro Ile Pro Ser Asn --NH.sub.2                                                       24) Lys Ala Ile His Ser Ser                                                  Asn Leu Pro Ile Pro Pro Asn                                                   --NH.sub.2                          25) Lys Ala Ile His Ser Ser Asn Phe Pro Ile Pro Pro Asn --NH.sub.2                                                       26) Hydrogen Ala Ile His Ser                                                 Ser Asn Leu Pro Ile Pro Pro                                                   Asn --NH.sub.2                      27) Lys Ala Ile Arg Ser Ser Asn Leu Ala Ile Ser Ser Asn --NH.sub.2                                                       28) Lys Ala Ile Arg Ser Ser                                                  Asn Leu Ala Val Ser Pro Asn                                                   --NH.sub.2                          29) Lys Ala Ile Arg Ser Ser Asn Leu Pro Val Pro Pro Asn --NH.sub.2                                                       30) Lys Thr Val His Ser Ser                                                  His Leu Ala Ala Leu Pro Asp                                                   --NH.sub.2                          31) Lys Thr Val His Ser Ser His Leu Ala Ala Ser Pro Asp --NH.sub.2                                                       32) Hydrogen Thr Val His Ser                                                 Ser His Leu Ala Ala Pro Ser                                                   Asp --NH.sub.2                      33) Lys Thr Val Arg Ser Ser His Leu Ala Ala Ser Pro Asp --NH.sub.2                                                       34) Lys Thr Val Arg Ser Ser                                                  His Leu Ala Ile Pro Pro Asp                                                   --NH.sub.2                          35) Lys Thr Val Arg Ser Ser His Leu Pro Ala Pro Pro Asp --NH.sub.2          __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 41                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 1:                          - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acids                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Ile Arg Ser Ser Asn Asn Leu Gly Ala Ile Le - #u Ser Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Thr Ser Asn Asn Leu Gly Ala Ile Le - #u Ser Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Leu Gly Pro Val Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Asn Asn Asn Leu Gly Pro Val Le - #u Ser Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser His Asn Leu Gly Ala Ala Le - #u Leu Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Phe Gly Ala Ile Leu Se - #r Ser Thr Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Pro Ile Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Phe Gly Pro Ile Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Arg Ser Asn Asn Phe Gly Pro Ile Leu Pr - #o Ser Thr Asn Val                      20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                                  35                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Pro Val Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Phe Gly Pro Ile Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pr - #o Pro Ser Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pr - #o Pro Ser Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Leu Gly Pro Val Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Leu Gly Pro Val Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Leu Gly Pro Val Leu Pr - #o Ser Thr Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Leu Gly Pro Val Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Leu Gly Pro Ile Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser Asn Asn Leu Gly Pro Ile Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Ile His Ser Ser Asn Asn Leu Gly Pro Ile Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val Ile Ser Ser Asn Asn Phe Gly Pro Ile Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Ile      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Leu Gly Pro Ile Leu Pr - #o Pro Thr Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Ile Arg Ser Ser Asn Asn Leu Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Ile Arg Ser Ser Asn Asn Leu Gly Ala Val Le - #u Ser Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Ile Arg Ser Ser Asn Asn Leu Gly Pro Val Le - #u Pro Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser His Asn Leu Gly Ala Ala Le - #u Leu Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser His Asn Leu Gly Ala Ala Le - #u Ser Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Thr Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser His Asn Leu Gly Ala Val Leu Pr - #o Ser Thr Asp Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser His Asn Leu Gly Ala Ala Le - #u Ser Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser His Asn Leu Gly Ala Ile Le - #u Pro Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Thr Asn Phe Leu      1               5   - #                10  - #                15               - - Val Arg Ser Ser His Asn Leu Gly Pro Ala Le - #u Pro Pro Thr Asp Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #cyclo linkage between the Asp                            and Lys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Lys Asp Asn Thr Ala Thr Lys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Ala Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Ser Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Pro Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Pro Ile Le - #u Pro Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pr - #o Ser Thr Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 1,6                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Le - #u Ala Asn Phe Leu Val      1               5   - #                10  - #                15               - - His Ser Ser Asn Asn Phe Gly Pro Val Leu Pr - #o Pro Ser Asn Val Gly                  20      - #            25      - #            30                   - - Ser Asn Thr Tyr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (B) LOCATION: 2,7                                                             (D) OTHER INFORMATION: - #disulfide bridge between                                 the Cys - #residues                                                      (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #Arg is a D amino acid residue                       (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #amidated Tyr (Tyrosinamide)                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Ar - #g Leu Ala Asn Phe Leu      1               5   - #                10  - #                15               - - Val His Ser Ser Asn Asn Phe Gly Ala Ile Le - #u Ser Ser Thr Asn Val                  20      - #            25      - #            30                   - - Gly Ser Asn Thr Tyr                                                              35                                                                   __________________________________________________________________________

We claim:
 1. A pharmaceutically acceptable salt of the peptide ##STR1##said peptide having an intramolecular linkage between the cysteine aminoacid residues at positions two and seven and tyrosinamide as thethirty-seventh amino acid residue.
 2. A composition comprising apharmaceutically acceptable salt of the peptide ##STR2## said peptidehaving an intramolecular linkage between the cysteine amino acidresidues at positions two and seven and tyrosinamide as thethirty-seventh amino acid residue, in a pharmaceutically acceptablecarrier.
 3. A composition comprising an acetate salt of the peptide##STR3## said peptide having an intramolecular linkage between thecysteine amino acid residues at positions two and seven and tyrosinamideas the thirty-seventh amino acid residue, in a pharmaceuticallyacceptable carrier.
 4. A composition comprising a hydrochloride salt ofthe peptide ##STR4## said peptide having an intramolecular linkagebetween the cysteine amino acid residues at positions two and seven andtyrosinamide as the thirty-seventh amino acid residue, in apharmaceutically acceptable carrier.
 5. A composition comprising atrifluoroacetate salt of the peptide ##STR5## said peptide having anintramolecular linkage between the cysteine amino acid residues atpositions two and seven and tyrosinamide as the thirty-seventh aminoacid residue, in a pharmaceutically acceptable carrier.